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Protein kinase R reveals an evolutionary model for defeating viral mimicry.

Elde NC, Child SJ, Geballe AP, Malik HS - Nature (2008)

Bottom Line: Distinguishing self from non-self is a fundamental biological challenge.Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry.We find that PKR has evolved under intense episodes of positive selection in primates.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

ABSTRACT
Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2alpha, which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2alpha) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2alpha recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular 'arms races' with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.

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PKR chimeras reveal masking of K3L sensitivity by Leu394(a) Ten-fold serial dilutions of transformants expressing alleles of human PKR with combinations of substitutions in the αE and αG helices are shown along with corresponding immunoblot analysis.(b) Phenotype ‘cubes’ summarizing the K3L susceptibility of alleles with all combinations of substitutions between human and gibbon PKR at positions 394, 489, and 492 from Figures 2a, 3a and S5. Red and blue dots indicate resistance and sensitivity to K3L respectively. With the exception of F-F-A, which shows some measure of resistance to K3L in the human background (indicated by the red crescent), each set of substitutions have similar phenotypes in the human and gibbon backgrounds. Each single substitution in wildtype human PKR results in a variant still resistant to K3L, while in two of three cases gibbon PKR becomes resistant (indicated by arrows).(c) Sequence alignments of the αG helix for each member of the eIF2α kinase family from several mammals highlights the conservation of this region compared to rapid evolution of PKR (black arrowheads indicate residues of the αG helix under positive selection in PKR). The frequency of substitutions among the panel at each position is indicated by a color code (yellow for a single substitution, orange for a second, red for a third, and blue for a fourth) with human sequence as a reference. Residues making contacts with eIF2α are indicated with lines below the PKR alignment.
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Figure 3: PKR chimeras reveal masking of K3L sensitivity by Leu394(a) Ten-fold serial dilutions of transformants expressing alleles of human PKR with combinations of substitutions in the αE and αG helices are shown along with corresponding immunoblot analysis.(b) Phenotype ‘cubes’ summarizing the K3L susceptibility of alleles with all combinations of substitutions between human and gibbon PKR at positions 394, 489, and 492 from Figures 2a, 3a and S5. Red and blue dots indicate resistance and sensitivity to K3L respectively. With the exception of F-F-A, which shows some measure of resistance to K3L in the human background (indicated by the red crescent), each set of substitutions have similar phenotypes in the human and gibbon backgrounds. Each single substitution in wildtype human PKR results in a variant still resistant to K3L, while in two of three cases gibbon PKR becomes resistant (indicated by arrows).(c) Sequence alignments of the αG helix for each member of the eIF2α kinase family from several mammals highlights the conservation of this region compared to rapid evolution of PKR (black arrowheads indicate residues of the αG helix under positive selection in PKR). The frequency of substitutions among the panel at each position is indicated by a color code (yellow for a single substitution, orange for a second, red for a third, and blue for a fourth) with human sequence as a reference. Residues making contacts with eIF2α are indicated with lines below the PKR alignment.

Mentions: Our analyses suggested that human PKR contained residues associated with increased resistance to K3L from both αG and αE helices. Indeed, we found that a human PKR allele carrying ‘susceptible’ mutations in both its αE (L394F) and αG (F489Y/S492A) helices loses wildtype resistance to K3L (Figure 3a, row 5). We tested all combinations of resistant and susceptible substitutions at positions 394 (helix αE), 489 and 492 (helix αG) in human PKR and found that six out of eight combinations of human PKR alleles resist K3L. The two exceptions are F-Y-A (described above) and F-F-A (Figure 3a, row 4), which is only slightly more resistant to K3L than F-Y-A, revealing a weak effect associated with the positively selected residue at position 489. While the human and gibbon PKR backbones bear similar outcomes at all positions (Figures 3b), the ‘susceptible’ human alleles still appear more resistant to vaccinia K3L than the ‘susceptible’ gibbon alleles, hinting at an additional K3L resistance determinant in the human PKR sequence (data not shown).


Protein kinase R reveals an evolutionary model for defeating viral mimicry.

Elde NC, Child SJ, Geballe AP, Malik HS - Nature (2008)

PKR chimeras reveal masking of K3L sensitivity by Leu394(a) Ten-fold serial dilutions of transformants expressing alleles of human PKR with combinations of substitutions in the αE and αG helices are shown along with corresponding immunoblot analysis.(b) Phenotype ‘cubes’ summarizing the K3L susceptibility of alleles with all combinations of substitutions between human and gibbon PKR at positions 394, 489, and 492 from Figures 2a, 3a and S5. Red and blue dots indicate resistance and sensitivity to K3L respectively. With the exception of F-F-A, which shows some measure of resistance to K3L in the human background (indicated by the red crescent), each set of substitutions have similar phenotypes in the human and gibbon backgrounds. Each single substitution in wildtype human PKR results in a variant still resistant to K3L, while in two of three cases gibbon PKR becomes resistant (indicated by arrows).(c) Sequence alignments of the αG helix for each member of the eIF2α kinase family from several mammals highlights the conservation of this region compared to rapid evolution of PKR (black arrowheads indicate residues of the αG helix under positive selection in PKR). The frequency of substitutions among the panel at each position is indicated by a color code (yellow for a single substitution, orange for a second, red for a third, and blue for a fourth) with human sequence as a reference. Residues making contacts with eIF2α are indicated with lines below the PKR alignment.
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Related In: Results  -  Collection

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Figure 3: PKR chimeras reveal masking of K3L sensitivity by Leu394(a) Ten-fold serial dilutions of transformants expressing alleles of human PKR with combinations of substitutions in the αE and αG helices are shown along with corresponding immunoblot analysis.(b) Phenotype ‘cubes’ summarizing the K3L susceptibility of alleles with all combinations of substitutions between human and gibbon PKR at positions 394, 489, and 492 from Figures 2a, 3a and S5. Red and blue dots indicate resistance and sensitivity to K3L respectively. With the exception of F-F-A, which shows some measure of resistance to K3L in the human background (indicated by the red crescent), each set of substitutions have similar phenotypes in the human and gibbon backgrounds. Each single substitution in wildtype human PKR results in a variant still resistant to K3L, while in two of three cases gibbon PKR becomes resistant (indicated by arrows).(c) Sequence alignments of the αG helix for each member of the eIF2α kinase family from several mammals highlights the conservation of this region compared to rapid evolution of PKR (black arrowheads indicate residues of the αG helix under positive selection in PKR). The frequency of substitutions among the panel at each position is indicated by a color code (yellow for a single substitution, orange for a second, red for a third, and blue for a fourth) with human sequence as a reference. Residues making contacts with eIF2α are indicated with lines below the PKR alignment.
Mentions: Our analyses suggested that human PKR contained residues associated with increased resistance to K3L from both αG and αE helices. Indeed, we found that a human PKR allele carrying ‘susceptible’ mutations in both its αE (L394F) and αG (F489Y/S492A) helices loses wildtype resistance to K3L (Figure 3a, row 5). We tested all combinations of resistant and susceptible substitutions at positions 394 (helix αE), 489 and 492 (helix αG) in human PKR and found that six out of eight combinations of human PKR alleles resist K3L. The two exceptions are F-Y-A (described above) and F-F-A (Figure 3a, row 4), which is only slightly more resistant to K3L than F-Y-A, revealing a weak effect associated with the positively selected residue at position 489. While the human and gibbon PKR backbones bear similar outcomes at all positions (Figures 3b), the ‘susceptible’ human alleles still appear more resistant to vaccinia K3L than the ‘susceptible’ gibbon alleles, hinting at an additional K3L resistance determinant in the human PKR sequence (data not shown).

Bottom Line: Distinguishing self from non-self is a fundamental biological challenge.Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry.We find that PKR has evolved under intense episodes of positive selection in primates.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

ABSTRACT
Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2alpha, which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2alpha) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2alpha recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular 'arms races' with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.

Show MeSH
Related in: MedlinePlus